FOXM1 在人胚胎干细胞基因组中的结合位点研究

作为转录因子 FOX 家族的成员， FOXM1 在细胞周期调控、细胞增殖分化、组织稳态和多种信号通路的调控等生物学过程发挥重要作用。在小鼠胚胎干细胞中发现， FOXM1可以调控细胞干性相关的转录因子 OCT4来维持小鼠胚胎干细胞的干性；同样在人类肿瘤干细胞中发现， FOXM1是 OCT4表达所必备的，表明FOXM1 在人类胚胎干细胞中具有潜在的作用。尽管近些年的研究发现， FOXM1参与胚胎干细胞的多能性和自我更新特征的维持，但是其在胚胎干细胞中的详细功能机制仍然不清楚。因此我们下载并分析了公共数据库的 FOXM1 的 ChIP-seq 数据，通过比较3种细胞系，我们发现 FOXM1 在他们中相同的结合位点非常少， 仅有 281 个， 并且 motif 预测的转录因子也并非 FOX 家族。因此我们选择染色质免疫沉淀后测序（ChIP-seq）这一应用广泛的研究 DNA-蛋白质互作的技术，在人类胚胎干细胞中进行探索。通过完善和优化在人类胚胎干细胞中的 ChIP-seq 实验流程， ChIP-seq实验结果表明，通过 FOXM1 抗体进行 ChIP-seq 在胚胎干细胞中找到的 FOXM1结合位点有限， 仅有两三百个， 不足以挖掘到有效的新的信息。由此，我们利用CRISPR-Cas9 技术，在 FOXM1 基因 3’末端整合了表位标签 FLAG 序列和新霉素
基因序列，成功构建了可以表达带 FLAG 标签的 FOXM1 蛋白和可以使用新霉素进行筛选的 pETChH1 细胞系，用于后续进一步研究。
 

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